Vol. 114: 69–75, 2015 DISEASES OF AQUATIC ORGANISMS Published May 11 doi: 10.3354/dao02851 Dis Aquat Org NOTE Chytridiomycosis in dwarf African frogs Hymenochirus curtipes B. G. Murphy1,*, C. Hillman1, J. M. Groff2 1Department of Pathology, Microbiology and Immunology and 2Pathology Service, The William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis, CA 95616-5270, USA ABSTRACT: Chytridiomycosis, resulting from an infection with the fungal agent Batracho - chytrium dendrobatidis (Bd), has resulted in widespread population declines in both wild and cap- tive amphibians. The dwarf African frog (DAF) Hymenochirus curtipes is native to central Africa and is commonly sold throughout North America as an aquarium pet species. Here we document fatal chytridiomycosis resulting from cutaneous Bd infections in DAF purchased directly from a pet store and from a historical lethal epizootic occurring at an aquaculture facility in central Cali- fornia, USA, more than 25 yr ago. Histological lesions and PCR-amplified sequence data were consistent with the etiology of Bd. The potential epidemiological relevance of this infection in DAF is discussed. KEY WORDS: Frog · Batrachochytrium dendrobatidis · Bd · Hymenochirus · Chytridiomycosis Resale or republication not permitted without written consent of the publisher INTRODUCTION (Parker et al. 2002). The pathogenesis, diagnosis and treatment of Bd-infected amphibians have recently Chytridiomycosis, resulting from an infection with been reviewed (Baitchman & Pessier 2013). the chytridiomycete fungus Batrachochytrium den- The relatively sudden appearance of chytridiomy- drobatidis (Bd), has caused dramatic declines in cosis can be explained by the hypothesis that Bd was amphibian populations around the world. Chytrid- recently introduced into new regions of the world, iomycota are heterotrophic fungi that are ubiquitous facilitating the infection of new amphibian species and cosmopolitan (Sparrow 1960, Karling 1977). (Daszak et al. 1999). Bd is a common infectious agent Chytridiomycota have been demonstrated to main- in African frogs from Ghana, Kenya and South Africa tain infectivity for up to 7 wk in lake water (Johnson (Weldon et al. 2004). African frogs that have been et al. 2003). Bd is found in both soil and water, acting widely exported from Africa include members of the as a primary saprobe using substrates such as chitin, Pipidae family, specifically, the African clawed frog keratin and plant detritus (Berger et al. 1998). At (ACF) Xenopus spp. and the dwarf African frog least 3 amphibian orders, Anura, Caudata and Gym - (DAF) Hymenochirus spp. (Hey 1986). Xenopus spp. nophiona, have been diagnosed with chytridiomyco- were widely exported starting in the 1930s for use in sis (Boyle et al. 2004, Churgin et al. 2013). Bd has pregnancy diagnosis, as laboratory animal models been identified in zoo collections (Pessier et al. 1999), and as aquarium pets, while Hymenochirus spp. the pet trade (Mutschmann et al. 2000), the food were disseminated widely in the aquarium pet trade. trade (Mazzoni 2003) and the laboratory animal trade It has been proposed that international trade of ACF *Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 70 Dis Aquat Org 114: 69–75, 2015 during the 20th century resulted in worldwide dis- 1991). In this report, the causative my cotic agent was semination of the chytridiomycete fungal agent (Wel- originally identified as Basidiobolus ranarum. Here, don et al. 2004). Multiple states in the USA now based upon lesion morphology (cutaneous hyperker- require a permit to purchase ACF. atosis), characteristic fungal thalli and new sequen- ACF (X. laevis) in the wild do not demonstrate clin- cing data, we more accurately define the causative ical signs of chytridiomycosis, and sudden die-offs of outbreak etiology as Bd. ACF have not been documented (Weldon et al. 2004). However, subclinical infections with Bd have been well documented in captive Xenopus laevis (Reed et MATERIALS AND METHODS al. 2000). In contrast, Bd infection in the related ACF X. tropicalis can result in anorexia and lethargy, cu- Animals taneous pigment changes, loss of the cutaneous slime layer and death (Parker et al. 2002). In a survey of Two DAF (Hymenochirus curtipes, frogs A and B, archived specimens of 3 different species of Xenopus Table 1) were purchased from a local pet store in from 1879 to 1999, the earliest case of chytridiomyco- 2010 and maintained in a small, established 7 l sis was found in a Xenopus laevis frog in 1938 (Wel- aquarium with locally obtained aquatic plants (horn- don et al. 2004). This fungal pathogen apparently ex- wort Ceratophyllum demursum), aquatic snails, de- isted as a stable endemic infection in southern Africa ionized water and gravel substrate. The frogs were for at least 23 yr before any Bd-infected specimens found dead approximately 1 wk after purchase and ap peared outside of Africa (Weldon et al. 2004). were immediately immersed in 10% buffered forma- Dwarf African frogs are small aquatic frogs native lin. Other than anorexia, clinical signs of morbidity to central Africa that lack both teeth and a tongue were not evident perimortem. and are completely aquatic. Otto Boettger, one of the From 1985 to 1987, 5 submissions for a total of ap- dwarf African frog discoverers, originally named proximately 75 post-metamorphic DAF (H. curtipes), them African dwarf clawed frogs in recognition of the approxima tely 0.25 g in weight, were submitted to the small black claws on their hind legs. Since these Fish Pathology Laboratory at the School of Veterinary claws are often lost as the frogs mature, these ani- Medicine, University of California, Davis, from a pri- mals are now more commonly re ferred to as dwarf vate culture facility in central California (Groff et al. African frogs. There are currently 4 recognized spe- 1991). The culturist reported a sustained increased cies of Hymenochirus: H. boulengeri, H. curtipes, mortality of post-metamorphic frogs that often ap - H. boettgeri, and H. feae (IUCN Red List of Threat- proached 100%, with a cumulative total of approxi- ened Species, www.iucnredlist.org). Currently, there is some controversy over the taxonomic Table 1. Details and sources of individual specimens of dwarf status of H. feae. Both H. boettgeri and H. cur- African frogs Hymenochirus curtipes described in this study. For tipes are commonly sold in the pet trade. As the histologic lesions, ‘+++’ indicates severe while ‘+’ indicates these animals look very similar to one another, mild severity. For PCR, ‘+’ indicates a successful amplification while ‘–’ indicates an unsuccessful amplification. Clones refers the morphological separation between these 2 to PCR amplicons that were subsequently cloned into plasmids, species can be challenging. In addition, these amplified in bacteria and sequenced frogs are capable of interbreeding and forming hy brids; as a result, some breeders feel that the Frog Source (year acquired) Histologic PCR Clones DAF sold in the pet trade are actually a ID lesions H. boettgeri × H. curtipes hybrid. The peer-reviewed literature has a single A Pet store (2010) +++ + 2 B Pet store (2010) +++ + 2 previous report of chytridiomycosis in DAF (H. C Direct from culturist (1985) + − boett geri) (Raverty & Reynolds 2001). Here we D Direct from culturist (1986) + − document fatal chytridiomycosis lesions and E Experimental infection (1987) + + 2 PCR-amplified and cloned Bd sequences iso- F Experimental infection (1987) + + 2 G Experimental infection (1987) + + lated from 2 H. curtipes individuals directly H Experimental infection (1987) + + purchased from a pet store. In addition, our I Experimental infection (1987) + + group re-evaluated the etiology of a published J Experimental infection (1987) + − fatal cutaneous mycotic epizootic in H. curtipes K Experimental infection (1987) + − L Experimental infection (1987) + − from a central California aquaculture facility M Experimental infection (1987) + − that occurred from 1985 to 1987 (Groff et al. Murphy et al.: Bd in dwarf African frogs 71 mately 10 000 frogs. Affected sentinel frogs (1985 and PCR 1986) exhibited a preference for the terrestrial envi- ronment rather than their normal aquatic environment Standard PCR was performed with DNA isolated a few days prior to death. External examination of from all 13 DAF (A−M). Standard PCR was per- dead sentinel frogs (Frogs C and D, Table 1) was unre- formed with Taq DNA polymerase (Invitrogen) on markable except for a slight pallor of the integument. an Eppendorf Mastercycler. Primers utilized for In 1987, an attempt was made to experimentally amplifying the 5.8s rRNA gene from Bd were as transmit the etiologic agent to naive DAF. Two follows: BATfor 5’-CCT TGA TAT AAT ACA GTG moribund-infected sentinel frogs (different animals TGC and BATrev 5’-AGC CAA GAG ATC CGT from Frogs C and D) and 12 healthy uninfected con- TGT CAA (Boyle et al. 2004). The primers were trol frogs were co-incubated together in a 20 l aquar- used in the following cycling conditions: 95°C for ium for 10 d at 20°C. The aquaria received well water 2 min followed by 40 cycles of 95°C for 15 s, 56°C at a flow rate of 0.1 l min−1. A control group of 12 for 30 s, 72°C for 30 s, followed by a final healthy frogs was maintained in similar conditions elongation step at 72°C for 5 min. PCR amplicons without ex posure to moribund frogs. After 10 d, 9 were analyzed on 2% agarose/ethidium bromide DAF be came moribund during this experimental gels; the predicted amplicon size was 146 bp. After transmission (Frogs E−M, Table 1). Moribund ani- the initial successful PCR with DNA extracted from mals were humanely euthanized with an overdose of Frogs A and B, DNA from one of these animals 500 mg tricaine methanesulfonate (MS-222) per ml of served as a positive control in subsequent PCR. A water. Postmortem, a midline incision was made in negative control sample (water template) was run the coelomic cavity and the frogs were immediately in parallel each time that the PCR was perfor med.